大量表現輔助蛋白及促進細胞循環以提升農桿菌轉殖水稻效率

Abstract

農桿菌轉殖法是目前普遍且重要的轉殖工具。此轉殖法主要優點為轉入的基因套數少甚至單一，不易引發基因靜默。然而除了多數模式植物外，在許多重要經濟作物上其轉殖效率仍偏低。農桿菌T-DNA在植物細胞中轉移的過程需藉由促轉基因轉譯之輔助蛋白參與協助。本試驗室先前透過共轉殖兩個T-DNA，一攜帶ET基因和另一GUS報告基因，並藉由GUS蛋白的活性強度來代表T-DNA的轉殖效率，已找出在水稻中包括BTI、VIP1、VirE3等幾個促轉基因能夠提升轉殖效率。因此本研究針對促轉基因載體的各個環節進行修改，以期達到進一步優化促轉基因的目的。首先為使促轉基因質體及報導基因質體以同一農桿菌共同攜帶，進行抗生素抗性測試並選出四環黴素抗性基因，作為報導基因之農桿菌專用篩選標誌。其次為增加促轉基因的表現，以四個候選啟動子進行啟動子強度測試，找出最強的啟動子2x35S-U。再者，為避免促轉基因插入植物後影響轉殖株的篩選，因此將促轉基因質體上植物篩選標誌的潮黴素抗性基因移除。接著，為抑制植物對T-DNA引發基因靜默，進行基因靜默抑制子的抑制強度測試，並選出P19抑制子。最後，構築一去除植物篩選標誌並帶有2x35S-U啟動子和P19抑制子的載體，進一步將各候選促轉基因裝載於該載體上，以進行促轉效率測試。結果發現BTI具有最佳的促轉效率。此外，RepA基因透過其編碼之蛋白，抑制RB蛋白並啟動細胞週期的G1期進入S期，本試驗亦作為促轉基因進行測試，然而並沒有觀察到轉殖效率的提升。

Agrobacterium-mediated T-DNA transfer is well-known to be a powerful plant transformation tool. This method mostly confer single to low-copies of integrated gene, therefore leads to less problems in gene-silencing. Although many model plants can be transformed by Agrobacterium, a lot of economically important crops are still “recalcitrant” to transformation. We therefore aim to improve plant transformation efficiency using rice as a test system. Accessory proteins, encoded by enhance transformation (ET) genes, are plant factors that involve in T-DNA transfer process within plant cell. By co-transformation of two Agrobacterium horbring two T-DNAs, one carry the ET gene and the other harbor the GUS reporter gene, expression of GUS enzyme was increased in presence of several ET genes, indicating an increase of T-DNA transfer efficiency. In this study, several modifications were employed to optimize the efficiency of ET gene. Firstly, to develop vector that can cohabit with the reporter-carrying vector in the same Agrobacterium, several antibiotics were examined and tetracycline resistance gene was chosen. Secondly, to increase expressions of ET genes, four promoters were tested and 2X35S-U was found to exhibit the strongest activity in rice. Thirdly, to decrease the selection preference of ET gene so to avoid its co-integration with target gene, plant hygromycin selection marker was eliminated from vector that harbors the ET gene. Fourthly, to inhibit gene-silencing effect of plant on the incoming T-DNA, suppressors borrowed from various viruses were screened and P19 was chosen as it confers the highest GUS expressions. Finally, a vector that contains 2X35S-U promoter and P19 viral suppressor, but not the plant selection marker, was constructed to host expressions of various ET genes. In the preliminary test using the constructed vector, BTI exhibited the best effect on enhance transformation. Moreover, a RepA gene that may promote cell cycles, presumably by inhibiting RB protein which blocks G1/S transition, was tested as an ET gene. However, no increase of transformation efficiency was observed.